OPA1 and MICOS Regulate Mitochondrial Crista Dynamics And

Hu et al. Cell Death and Disease (2020) 11:940 https://doi.org/10.1038/s41419-020-03152-y Cell Death & Disease

ARTICLE Open Access OPA1 and MICOS Regulate mitochondrial crista dynamics and formation Chao Hu1,LiShu1, Xiaoshuai Huang2, Jianglong Yu1, liuju Li2, Longlong Gong1, Meigui Yang1, Zhida Wu1, Zhi Gao1, Yungang Zhao3, Liangyi Chen2 and Zhiyin Song1

Abstract Mitochondrial cristae are the main site for oxidative phosphorylation, which is critical for cellular energy production. Upon different physiological or pathological stresses, mitochondrial cristae undergo remodeling to reprogram mitochondrial function. However, how mitochondrial cristae are formed, maintained, and remolded is still largely unknown due to the technical challenges of tracking mitochondrial crista dynamics in living cells. Here, using live-cell Hessian structured illumination microscopy combined with transmission electron microscopy, focused ion beam/ scanning electron microscopy, and three-dimensional tomographic reconstruction, we show, in living cells, that mitochondrial cristae are highly dynamic and undergo morphological changes, including elongation, shortening, fusion, division, and detachment from the mitochondrial inner boundary membrane (IBM). In addition, we ﬁnd that OPA1, Yme1L, MICOS, and Sam50, along with the newly identiﬁed crista regulator ATAD3A, control mitochondrial crista dynamics. Furthermore, we discover two new types of mitochondrial crista in dysfunctional mitochondria, “cut- through crista” and “spherical crista”, which are formed due to incomplete mitochondrial fusion and dysfunction of the MICOS complex. Interestingly, cut-through crista can convert to “lamellar crista”. Overall, we provide a direct link between mitochondrial crista formation and mitochondrial crista dynamics. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,;

Introduction establish and maintain their unique structure to provide a Mitochondria contain outer and inner membranes. The large amount of surface area for chemical reactions to inner mitochondrial membrane is organized in two occur. Mitochondrial cristae have an orderly arrangement morphologically distinct regions: the inner boundary and contain respiratory chain supercomplexes, cyto- membrane (IBM) and the crista membrane (CM)1. The chrome c, and certain proteins that regulate mitochon- IBM contains translocases and certain proteins, such as drial oxidative phosphorylation and electron transfer3,4. OXA1 and Mia40, to shuttle proteins into the mito- Three-dimensional (3D) images show that mitochondrial chondrial matrix2. The CM is usually connected to IBM cristae are bag-like structures1,5. by the crista junction (CJ), which is a narrow constric- Mitochondrial cristae have long been considered tubu- tion3,4. Mitochondrial cristae harbor proteins that lar or lamellar invaginations formed by protrusion of the IBM into the mitochondrial matrix. OPA1, dimeric F1FO- ATP synthase, and MICOS (mitochondrial contact site Correspondence: Liangyi Chen ([email protected])or Zhiyin Song ([email protected]) and cristae organizing system) complex play key roles – 1Hubei Key Laboratory of Cell Homeostasis, Frontier Science Center for during this process6 8. OPA1 oligomerization narrows the Immunology and Metabolism, College of Life Sciences, Wuhan University, CJ and promotes ATP synthase dimerization9,10. The Wuhan, Hubei 430072, China 2State Key Laboratory of Membrane Biology, Beijing Key Laboratory of dimerization of ATP synthase is necessary to control Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking crista biogenesis and morphology, and it induces a posi- University, Beijing, China tive curvature that results in the formation of a crista Full list of author information is available at the end of the article 6 These authors contributed equally: Chao Hu, Li Shu sheet at the tip . The MICOS complex regulates the Edited by M. Campanella

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formation of CJs and mediates the contact between the study the dynamic changes in mitochondrial cristae in living OMM and IMM by interacting with the SAM (the sorting cells. We found that the membrane components of the and assembly machinery) complex, which is located on mitochondrial IBM and cristae membrane were not evenly the mitochondrial outer membrane and functions in the distributed within the mitochondria (Figs. S1A–S1C). assembly of β-barrel proteins into the mitochondrial outer Therefore, communication and material exchange between membrane11,12. The MICOS complex consists of at least the mitochondrial cristae and IBM should occur to maintain 7 subunits in mammals: Mic60, Mic27, Mic26, Mic25, mitochondrial membrane homeostasis. In living cells, mito- Mic19, Mic13, and Mic103. Recently, Harner et al. chondrial cristae were mainly tubular or lamellar, highly hypothesized that mitochondrial fusion and the MICOS dynamic, and organized, with remarkable changes in length complex contribute to the formation of mitochondrial and position within one second (Fig. 1A–C and Supple- lamellar cristae7. mentary Video 1). The mitochondrial cristae constantly Under normal conditions, normal tubular or lamellar altered their lengths by elongation or shortening, detached mitochondrial cristae have an orderly arrangement. from the IBM, or contacted and fused with the IBM (Fig. However, under different physiological and pathological 1A–C and S1D), suggesting that mitochondrial cristae are conditions, the number, size, shape, and distribution of dynamically altered in length and constantly contact and mitochondrial cristae are affected to a very different communicate with the IBM. Moreover, two mitochondrial extent1,3,4,9,13. For example, in aged animals, mitochon- cristae could fuse into one (Fig. 1D), and one crista could drial cristae are enlarged, and ~25% of mitochondria divide into two (Fig. 1E). Occasionally, mitochondrial cristae contain large central voids that lack CJs14,15. However, the contacted each other and fused to form a crista network; mechanism and functions of mitochondrial crista remo- however, this contact was subsequently separated (Fig. 1F), deling are largely unclear. suggesting that mitochondrial cristae contact each other via a Mitochondria continuously fuse and divide to maintain “kiss-and-run” mode. We also measured the mitochondrial normal mitochondrial morphology and functions. Mitofusins crista movement rate and found that mitochondrial IBMs (Mfn1 and Mfn2) and OPA1 mediate outer and inner had a substantial overlap, but a few mitochondrial cristae did mitochondrial membrane fusion, respectively16,17. Addition- not, as seen by comparing the 0-sec and 0.1-sec images (Fig. ally, Drp1 is a key regulator of mitochondrial fission18,19. 1G). However, most mitochondrial cristae showed no over- Theoretically, similar to mitochondria, mitochondrial cristae lap between the 0-sec and 2-sec images (Fig. 1H). Addi- should be highly dynamic. However, due to the limitation of tionally, mitochondrial cristae can undergo elongation, technology in detecting mitochondrial cristae in living cells, shortening, detachment, fusion, and fission in one or a few mitochondrial crista dynamics are not fully understood. seconds (Fig. 1A–F). These data suggest that mitochondrial For more than half a century, mitochondrial cristae have cristae are highly dynamic and change within a few seconds. only been analyzed by transmission electron microscopy Mitochondria can contact each other by a “kiss-and-run” (TEM), which cannot be applied to living cells. Recently, mode28. We then investigated whether mitochondrial cristae the development of super-resolution microscopy tech- are involved in mitochondrion–mitochondrion contact by nology has provided an excellent tool for visualizing Hessian-SIM. During mitochondrion–mitochondrion con- – mitochondria or mitochondrial cristae in living cells20 26. tact (kiss-and-run fusion), direct mitochondrial crista–crista We previously established a super-resolution microscopy contact was observed (Fig. 2A). Moreover, during mito- technology called structured illumination microscopy chondrial fusion, mitochondrial cristae contact and fuse with (Hessian-SIM), which enables fast, long-term, and super- each other (Fig. 2B). Our data are consistent with a previous resolution imaging of mitochondrial cristae27. Here, we report indicating that the cristae of adjacent mitochondria used Hessian-SIM combined with TEM, focused ion are coordinated at inter-mitochondrial junctions in cardio- beam/scanning electron microscopy (FIB-SEM), and 3D myocytes29.Overall,thesefindingssuggestthatmitochon- tomographic reconstruction to analyze the dynamic drial cristae contact and communicate with each other within changes in mitochondrial cristae and to study the role of and between mitochondria. the related factors in regulating mitochondrial crista Due to the limitation of Hessian-SIM resolution, we shape, quantity, arrangement, and distribution. could not distinguish crista–IBM and crista-cristae fusion or contact; thus, we investigated mitochondrial crista Results morphology by TEM. The contact site between mito- Mitochondrial cristae are highly dynamic and chondrial cristae and the IBM is called the CJ. Based on communicate with each other the number of CJs per crista, mitochondrial crista could Hessian-SIM allows MitoTracker Green FM (specifically be classified into 3 categories: (1) type I cristae, mito- stains mitochondria independent of mitochondrial mem- chondrial crista containing one CJ; (2) type II cristae, brane potential)-labeled mitochondrial cristae to be visua- mitochondrial crista containing two CJs; and (3) type III lizedandtrackedinlivingcells27. We used Hessian-SIM to cristae, mitochondrial crista without CJ. Most

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Fig. 1 (See legend on next page.)

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(see ﬁgure on previous page) Fig. 1 Mitochondrial cristae dynamics in live cells. A Representative live-cell time-lapse images of mitochondria in HeLa cells. Mitochondria in HeLa cells were stained with MitoTracker Green (250 nM, 15 min), then were tracked by time-lapse Hessian-SIM (90 frames/sec), 9 raw frames were used to reconstruct one super-resolution image. The indicated images demonstrate the mitochondrial cristae can shorten or extension in a very short period of time. Green, red, and blue arrowheads indicate the dynamic cristae. B Representative live-cell time-lapse images of mitochondrial cristae detached from the inner boundary membrane (IBM) and subsequently shortening. HeLa cells were treated with MitoTracker Green (250 nM, 15 min) and tracked by time-lapse by Hessian-SIM as described in (A). The white arrows marked the continuous shortening of mitochondria cristae, respectively, the red arrows indicate the cristae detached from the mitochondrial IBM then shorten in the mitochondrial matrix. C Representative live- cell time-lapse images of mitochondrial cristae fusing with IBM. HeLa cells were treated with MitoTracker Green (250 nM, 15 min) and tracked by time- lapse Hessian-SIM as described in (A). The green arrowheads showed that the site of mitochondrial cristae fusing with the IBM. D Representative live-cell Hessian-SIM images of mitochondrial cristae fusion event. HeLa cells were treated with MitoTracker Green (250 nM, 15 min) and tracked by time-lapse Hessian-SIM as described in (A). The selected images show that two mitochondrial cristae fused into one crista, the green and red arrows mark the mitochondrial cristae fusion event. E Representative live-cell Hessian-SIM images of mitochondrial crista division event. HeLa cells were stained with MitoTracker Green (250 nM, 15 min) and tracked by time-lapse Hessian-SIM as described in (A). The green and red arrows indicate the mitochondrial crista division process within 3 s. F Representative live-cell time-lapse images of mitochondrial crista–crista contact. HeLa cells were treated with MitoTracker Green (250 nM, 15 min) and tracked by time-lapse Hessian-SIM as described in (A). The green and red arrowheads-indicated mitochondrial cristae contacted each other and then were separated. G, H HeLa cells were stained with MitoTracker Green (250 nM, 15 min) and tracked by time-lapse Hessian-SIM as described in (A). The selected two images (t = 0 s, 0.1 s) (G)or(t = 0 s, 2 s) (H) were merged to show mitochondrial crista dynamic change. The white arrowheads indicate the moved mitochondrial cristae.

mitochondrial cristae of HeLa or COS7 cells belong to cristae were straight, had an orderly distribution in the types I and II, and only a few cristae are type III (Fig. 3A, mitochondria and were nearly parallel to each other (Fig. B). Under normal conditions, the mitochondrial cristae 4A). Importantly, WT MEF mitochondrial cristae were are mainly type I, and a few type II and type III cristae highly dynamic, displaying shortening, elongation, contact exist in HeLa and HCT116 cells (Fig. 3C–F). Consistently, with IBM, etc. (Fig. 4B and Supplementary Video 2). In 3D tomographic reconstruction of mitochondria further OPA1 knockout (KO) MEFs, mitochondria were fragmented, confirmed the existence of type I, II, and III mitochondrial mitochondrial cristae were decreased, and crista dynamics, cristae (Fig. 3G). The prevailing view is that the IBM can including shortening, elongation, crista–IBM contact (or invaginate into the mitochondrial matrix to form mito- fusion), and crista–crista contact (or fusion), were markedly chondrial cristae6,30, and this theory explains the exis- decreased (Fig. 4A, B Supplementary Video 3), indicating that tence of type I mitochondrial cristae. However, the OPA1 regulates mitochondrial crista formation and existence of type III cristae indicates that mitochondrial dynamics. Interestingly, crista detachment from the IBM cristae are able to detach from the IBM. The type II (loss of CJs) was increased in OPA1 KO mitochondria (Fig. cristae, on the other hand, suggests that mitochondrial 4A, B and Supplementary Video 3), suggesting that OPA1 is cristae can fuse with the IBM. Then, we analyzed a large associated with the maintenance of CJs. Our findings are number of mitochondria and captured TEM images dis- consistent with a previous reportthatOPA1oligomerization playing contact and fusion between cristae and the IBM is involved in the maintenance of CJs32,indicatingthatOPA1 (Fig. 3H, I) or images representing the crista network (Fig. may form a “staple” across CJs. Consistently, TEM imaging 3J). These data further support the dynamic character- confirmed that OPA1 KO led to decreased mitochondrial istics of mitochondrial cristae. cristae and caused a relative increase in abnormal mito- Based on our findings, we propose a new understanding chondrial cristae that are crooked, are disordered, or contain of mitochondrial crista dynamics (Fig. 3K). In living cells, 0 or 2 crista junctions (Fig. 4CandS2A–S2C). Moreover, mitochondrial cristae are tubular and lamellar and highly OPA1 KO mitochondria displayed relatively increased CJs ordered in mitochondria; they are highly dynamic and per crista and increased crooked cristae compared with WT continuously elongated or shortened. Importantly, mito- mitochondria (Fig. 4CandS2D–S2E). Interestingly, type II chondrial crista dynamics are highly associated with crista cristae are relatively stable, displaying few crista shortening biogenesis: crista elongation is related to crista formation, and elongation events (Supplementary Video 3). crista–IBM contact and fusion can form CJs, and Yme1L is an inner mitochondrial membrane protease that detachment of crista from the IBM leads to the loss of CJs. cleaves OPA1 at the S2 site, and Yme1L depletion results in increased OMA1-mediated OPA1 processing at the S1 – OPA1 and Yme1L regulate mitochondrial crista formation, site33 36. Therefore, we investigated whether Yme1L regulates morphology, arrangement, and dynamics mitochondrial crista dynamics. Hessian-SIM and TEM ima- OPA1 is required for maintaining mitochondrial cristae31. ging revealed that Yme1L KO caused mitochondrial frag- We thus investigated the role of OPA1 in mitochondrial mentation and led to fewer mitochondrial cristae, more crista dynamics. In WT MEFs, most of the mitochondrial abnormal mitochondrial cristae, and more CJs per crista

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Fig. 2 Mitochondrial crista–crista contact between two mitochondria. A, B Representative live-cell time-lapse Hessian-SIM images of mitochondria in HeLa (A) or COS7 (B) cells stained with MitoTracker Green (250 nM, 15 min). Time-lapse Hessian-SIM images reveal that mitochondrial crista–crista contact between two mitochondria during mitochondrial fusion. The red arrows demonstrate crista–crista contact.

(Fig. 4D–FandS2F–S2I). Furthermore, most Yme1L KO large number of mitochondrial cristae were distributed mitochondrial cristae were crooked and arranged in a dis- parallel to the IBM, and a few mitochondrial cristae ordered manner (Fig. 4F and S2J). In addition, Yme1L KO exhibited an onion-like structure in Mic10 KO, Mic19 mitochondrial cristae, which were abnormal in shape, were KO, and Mic60 KD cells (Fig. 4G). Further time-lapse also highly dynamic and moved quickly, similar to the control Hessian-SIM showed that mitochondrial cristae were mitochondrial cristae, but the crista dynamic events of short- unevenly distributed in the mitochondria in Mic10 KO, ening and detachment from the IBM were markedly Mic19 KO and Mic60 KD cells, and the ﬂuorescence of decreased (Fig. 4E and Supplementary Videos 4 and 5), sug- cristae was stronger than that of the IBM (Fig. 4H). gesting that Yme1L is an important regulator of mitochondrial Moreover, in Mic10 KO, Mic19 KO, and Mic60 KD cells, crista dynamics. Interestingly, crista–IBM and crista–crista mitochondrial crista dynamic events, including short- contact (or fusion) events were remarkably increased in ening, elongation, and crista–crista contact (or fusion), Yme1L KO mitochondria compared with the control (Fig. 4E were remarkably decreased (Fig. 4I and Supplementary and Supplementary Videos 5), suggesting that Yme1L reg- Videos 6–8), suggesting that the MICOS complex regulates mitochondrial crista–crista communication. ulates mitochondrial crista dynamics. Additionally, the ﬂuorescence of Mic10 KO, Mic19 KO, and Mic60 KD The MICOS complex and its associated proteins regulate cristae did not diffuse into the IBM (Fig. 4H and Sup- mitochondrial crista junction formation, crista–IBM fusion, plementary Videos 6–8), indicating that Mic10 KO, and cristae distribution Mic19 KO, and Mic60 KD cristae do not fuse with the We assessed the role of MICOS complex subunits, IBM due to the loss of CJs. These results suggest that the including Mic10, Mic19, and Mic60, in the regulation of MICOS complex is essential for mitochondrial crista mitochondrial CJs. As seen by TEM analysis, Mic10 KO, junction formation and crista–IBM fusion. Additionally, Mic19 KO, or Mic60 knockdown (KD) resulted in a slight only a few mitochondrial cristae were observed by decrease in the number of mitochondrial cristae, an Hessian-SIM (Fig. 4H), although many cristae exist in increase in abnormal cristae, and a complete loss of Mic10 KO, Mic19 KO, and Mic60 KD mitochondria (Fig. mitochondrial CJs (Fig. 4G and S2K–S2P). Furthermore, a 4G), probably due to changes in the mitochondrial

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Fig. 3 (See legend on next page.)

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(see figure on previous page) Fig. 3 Mitochondrial cristae types and dynamic events. A, B HeLa (A) and COS-7 (B) cells were stained with MitoTracker Green (250 nM, 15 min), and then imaged by Hessian-SIM. The white arrows marked the mitochondrial cristae lacking crista junction. C–F Mitochondrial cristae in HeLa (C)or HCT116 (E) cells were analyzed and imaged by transmission electron microscopy (TEM), and the representative image of mitochondrial ultrastructure was drawn. The red numbers represent the different types of mitochondrial cristae morphology. “1” represents mitochondrial crista with one crista junction (CJ), “2” represents mitochondrial crista containing two CJs, “3” represents mitochondrial crista lacking CJs. Then, the number of HeLa (D)or HCT116 (F) mitochondrial cristae types were counted (three independent experiments, 100 mitochondria cristae for each experiment). G HeLa cells were fixed, filtrated, polymerized, and embedded in EPON 812 resin, the specimen of 70 nm sections was then imaged using Fei Tecnai Spirit Transmission Microscope. There-dimensional (3D) reconstruction and segmentation of mitochondria were completed using 3D-IMOD software. Cyan: mitochondrial crista containing CJ; red, mitochondrial crista lacking CJ. H–J HeLa mitochondrial cristae were analyzed and imaged by TEM, and the representative images of mitochondrial ultrastructure were drawn. The indicated mitochondrial cristae dynamic events were displayed, the red arrows in the drawing picture indicate the cristae contacting IBM (H), crista containing two CJs (I), or crista–crista contact site (J). K The models of mitochondrial cristae dynamics. 1. Mitochondrial cristae division: one crista splits into two independent cristae; 2. Mitochondrial cristae fusion: two cristae fused into one crista; 3. Mitochondrial cristae shorten: the length of cristae is shortened; 4. Mitochondrial growth and elongation: crista undergoes growth and elongation; 5. Mitochondrial crista detachment: crista is detached from IBM, meanwhile, lost a Cj; 6. Mitochondrial cristae network: cristae can fuse with each other to form a network structure. 7. Mitochondrial cristae communication inter-mitochondria: cristae contacts and communicates with each other between two mitochondria.

membrane organization or the composition of the inner regulating mitochondrial crista morphology. Additionally, mitochondrial membrane, which leads to little staining ATAD3A depletion remarkably reduced the levels of with MitoTrack Green22. OPA1, Yme1L, and Mic60 (Fig. S2T), suggesting that The Sam50-Mic19-Mic60 axis plays a key role in mito- ATAD3A may cooperate with OPA1, Yme1L and MICOS chondrial crista junction formation37. Hessian-SIM to regulate mitochondrial crista morphology. showed that Sam50-GFP signals were puncta that faced We also used actinomycin D (ActD), a potent inducer of CJs (Fig. S2Q–S2R). Thus, we investigated the effect of apoptosis, for physiological stimulation to analyze mito- Sam50 KD on mitochondrial cristae. Sam50 KD reduced chondrial crista morphology. ActD treatment induced the number of mitochondrial cristae and resulted in a rapid mitochondrial fission in HeLa cells, leading to a significant decrease in the number of CJs per mitochon- dramatically increased number of fragmented mitochondrial crista but an increased number of abnormal mito- dria (Figs. S3A–S3B). Moreover, ActD treatment reduced chondrial cristae (Fig. 4G and S2N–S2P). Moreover, in the number of mitochondrial cristae and significantly Sam50 KD cells, mitochondrial cristae were nearly parallel increased the number of abnormal mitochondrial cristae to the IBM (Fig. 4G), and mitochondrial crista dynamic (Figs. S3C–S3E). Additionally, the number of CJs per crista events, including elongation, shortening, and fusion with remained unchanged, but the number of crooked cristae IBM, were markedly decreased (Fig. 4I and Supplementary and the cristae width were increased in ActD-treated cells Video 9). Additionally, Sam50 KD impairs mitochondrial (Figs. S3C, S3F, and S3G). These data suggest that ActD membrane biogenesis and organization, which may affect treatment induces changes in mitochondrial crista shape. MitoTracker Green FM staining and imaging of Hessian- SIM in living Sam50 KD cells (Supplementary Video 9). Incomplete mitochondrial fusion contributes to the By co-IP and mass spectrometry analysis, we identified a formation of “cut-through crista” that are maintained by new MICOS-interacting protein, ATAD3A (Fig. S2S and the MICOS complex supplementary Table 1). ATAD3A KO resulted in Mitochondrial cristae are tethered to the IBM by CJs, abnormal crista morphology and reduced the number of which are narrow and ring-like or slot-like structures3,6.To mitochondrial cristae but led to a significant increase in furtherstudythemorphologyofCJs,weusedFIB-SEM the number of CJs per mitochondrial crista (Fig. 4G and analysis and 3D tomography to reconstruct the structure of S2N–S4P), indicating that ATAD3A is involved in main- mitochondrial cristae and CJs. In HeLa cells, most mito- taining CJ. Moreover, ATAD3A KO resulted in increased chondrial cristae are lamellar (Fig. 5A and Supplementary mitochondrial crista–IBM and crista–crista contact (Fig. Video11).Interestingly,HeLamitochondriacontainafew 4G–I and and Supplementary Video 10). Occasionally, the crista-like inner-membrane compartments that fully connect mitochondrial cristae network was found in ATAD3A KO the IBM and open to the inner membrane space (IMS) (Fig. HeLa cells (Fig. 4G). These data indicate that ATAD3A 5A, B and Supplementary Video 11). The distance (~30 nm) negatively regulates the contact and subsequent fusion of between the two membranes of this compartment is very mitochondrial cristae with the IBM or adjacent mito- similar to the distance between lamellar cristae (Fig. 5A). chondrial cristae. Therefore, mitochondrial crista–IBM Therefore, we named this inner-membrane compartment a and crista–crista contacts play an important role in cut-through crista. Cut-through crista does not contain a

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Fig. 4 (See legend on next page.)

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(see figure on previous page) Fig. 4 Mitochondrial cristae dynamics is regulated by OPA1, Yme1L, MICOS complex, and ATAD3A. A Representative Hessian-SIM images of mitochondria in Live WT and OPA1 knockout (KO) MEFs stained with MitoTracker Green (250 nM, 15 min). B Live WT and OPA1 KO MEFs were stained with MitoTracker Green (250 nM, 15 min), and tracked and imaged by time-laps Hessian-SIM. Mitochondrial cristae dynamic events, including shortening, elongation, crista–IBM contact, crista–crista contact, and crista detaching from IBM were quantified (three independent experiments, about 100 mitochondria cristae for each experiment, total mitochondria cristae were indicated). Statistical significance was assessed from the student’s t-test; error bars indicate the means ± SD of three independent experiments, N.S. indicates “not significant”, ***p < 0.001 versus control. C Representative TEM images of mitochondria in WT and OPA1 KO MEFs. D Representative Hessian-SIM images of mitochondria in live control and Yme1L KO HCT116 cells stained with MitoTracker Green (250 nM, 15 min). E Live control and Yme1L KO HCT116 cells were stained with MitoTracker Green (250 nM, 15 min), and tracked and imaged by time-laps Hessian-SIM. Mitochondrial cristae dynamic events, including shortening, elongation, crista–IBM contact, crista–crista contact, and crista detaching from IBM were quantified (three independent experiments, about 100 mitochondria cristae for each experiment, total mitochondria cristae were indicated). Statistical significance was assessed from the student’s t-test; error bars indicate the means ± SD of three independent experiments, N.S. indicates “not significant”,*p < 0.05, ***p < 0.001 versus control. F Representative TEM images of mitochondria in control and Yme1L KO HCT116 cells. G Representative TEM images of mitochondria in control, Mic10 KO, Mic19 KO, Mic60 knockdown (KD), Sam50 KD, and ATAD3A KO HeLa cells. Magnified images in the boxed regions are shown as insets. H Live control, Mic10 KO, Mic19 KO, Mic60 KD, Sam50 KD, and ATAD3A KO HeLa cells were stained with MitoTracker Green (250 nM, 15 min) and imaged by Hessian-SIM. The representative mitochondrial cristae were displayed. I Live control, Mic10 KO, Mic19 KO, Mic60 KD, Sam50 KD, and ATAD3A KO HeLa cells were stained with MitoTracker Green (250 nM, 15 min), and tracked and imaged by time-laps Hessian-SIM. Mitochondrial cristae dynamic events, including shortening, elongation, crista–IBM contact, crista–crista contact, and crista detaching from IBM were quantified (three independent experiments, about 100 mitochondria cristae for each experiment, total mitochondria cristae were indicated). Statistical significance was assessed from the student’s t-test; error bars indicate the means ± SD of three independent experiments, **p < 0.01, ***p < 0.001 versus control.

crista tip, and its CJ surround the edge of crista in a long during mitochondrial fusion in living cells was tracked by groove. We previously demonstrated that outer and inner Hessian-SIM. Normally, mitochondrial fusion does not mitochondrial membrane fusion are separate processes and lead to the formation of new mitochondrial cristae in that OPA1 mediates inner membrane fusion16. According to HeLa cells (Fig. S4A). However, sometimes, two mito- the morphological characteristics of cut-through crista, we chondria contact and fuse to form a cut-through crista, hypothesized that the formation is probably due to the lack probably due to loss of inner mitochondrial membrane of inner mitochondrial membrane fusion after outer mito- fusion (Fig. 5I and S4B). Additionally, approximately chondrial membrane fusion. We then analyzed the inner 10–15% of mitochondrial fusion leads to cut-through mitochondrial membrane structure in OPA1 KO cells lack- crista in WT cells. Interestingly, by tracking OPA1 KO ing inner mitochondrial membrane fusion. The Hessian-SIM mitochondria, we observed that certain cut-through crista assay revealed that some OPA1 KO mitochondria were (just one side) could detach from the IBM and form a closely connected together (the red arrow indicates the tether lamellar crista (Fig. 5J), suggesting that cut-through crista site) (Fig. 5C), and a cut-through crista (yellow arrowhead may be the preform of lamellar cristae. indicated) was also formed in OPA1 KO mitochondria (Fig. Together, according to our ﬁndings, we propose a mode 5C). Moreover, 3D tomographic reconstruction conﬁrmed of cut-through crista formation due to incomplete mito- the existence of a cut-through crista in the OPA1 KO chondrial fusion (Fig. S4C–S4D). mitochondrion, and OPA1 KO mitochondria had a larger proportion of cut-through crista than did WT mitochondria Spherical crista, a new type of mitochondrial crista, is formed (Fig. 5D–F and Supplementary Video 12), suggesting that due to incomplete mitochondrial fusion and loss of CJ OPA1 dysfunction promotes the formation of cut-through Mic10 is required for CJ formation. The TEM images crista. Overall, outer mitochondrial membrane fusion and displayed some onion-like cristae in Mic10 KO mito- the subsequent absence of inner mitochondrial membrane chondria (Fig. S5A). Surprisingly, FIB-SEM and 3D fusion induce the formation of cut-through crista. tomographic reconstruction of the onion-like cristae in We also investigated the role of the MICOS complex in the Mic10 KO mitochondrion formed a new type of cut-through crista formation by FIB-SEM, and 3D mitochondrial crista, a “spherical crista”, which is a tomographic reconstruction. Mic10, a key component of double-membrane sphere and lacks CJs (Fig. 6A–B and the MICOS complex, plays a central role in CJ formation Supplementary Video 14). Only 20–30 percent of the (Fig. 4G and S2P). In Mic10 KO mitochondria, cut- onion-like cristae displayed spherical crista after 3D through crista were not observed (Fig. 5G–H and Sup- tomographic reconstruction. Additionally, spherical crista plementary Video 13), indicating that the MICOS com- also existed in OPA1 KO mitochondria (Fig. 6C and plex is required for the maintenance of cut-through crista. Supplementary Video 15). These results demonstrate To directly study the process of cut-through crista that OPA1 or Mic10 depletion promotes the formation formation, inner mitochondrial membrane remodeling of spherical crista. Then, we investigated the effect of

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(See legend on next page.)

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(see figure on previous page) Fig. 5 Identification of mitochondrial “cut-through crista”.AFocused ion beam/scanning electron microscopy (FIB-SEM) image of mitochondria in HeLa cells. 3D reconstruction and segmentation of mitochondrion showing normal crista and cut-through crista from FIB-SEM were completed by using 3D-IMOD software. White, mitochondrial outer membrane; cyan, mitochondrial IBM and “cut-through crista”; purple red, lamellar crista. B Quantification of the “cut-through crista” in mitochondria of HeLa cells (n = 100 cristae). Error bars indicate the means ± SD of three independent experiments. C Live OPA1 KO MEFs were stained with MitoTrack Green and then imaged by Hessian-SIM. D The 70 nm section of specimen of HCT116 OPA1 KO cells was analyzed and imaged by Fei Tecnai Spirit Transmission Microscope. 3D reconstruction and segmentation of mitochondrion were completed using 3D-IMOD software. Blue, mitochondrial outer membrane; cyan, mitochondrial IBM and “cut-through crista”; green, onion-like crista; purple red, tubular crista. E Representative thin-section FIB-SEM images of mitochondrion showing cut-through crista with 2 crista junctions in HCT116 OPA1 KO cells. 3D tomographic reconstruction and segmentation of OPA1 KO mitochondrion from FIB-SEM were performed by using 3D-IMOD software. White, mitochondrial outer membrane; cyan, mitochondrial IBM and “cut-through crista”. F Quantification of the “cut-through crista” in mitochondria of control and OPA1 KO HCT116 cells (n = 100 cristae). “others” means “other types of crista except for cut- through crista”. Statistical significance was assessed from the student’s t-test; error bars indicate the means ± SD of three independent experiments, **p < 0.01 versus control. G The continuous 10 nm-thick sections (total 300 nm) from Mic10 KO HeLa cells were analyzed and imaged by FIB-SEM. 3D tomographic reconstruction and segmentation of mitochondrion from FIB-SEM were performed by 3D-IMOD software. White, mitochondrial outer membrane; cyan, mitochondrial IBM; purple red and yellow, lamellar crista without CJ. H Quantification of the “cut-through crista” in mitochondria of control and Mic10 KO HeLa cells. “others” means “other types of crista except for cut-through crista” (n = 100 cristae). Statistical significance was assessed from the student’s t-test; error bars indicate the means ± SD of three independent experiments, ***p < 0.001 versus control. I Mitochondria in COS-7 cells were stained with 250 nM MitoTracker Green for 15 min, then were tracked and imaged by time-lapse Hessian-SIM. The green arrowhead indicates the contact and fusion site of two different mitochondria, the red arrowhead indicates the newly formed crista. J Live OPA1 KO MEFs were stained with MitoTracker Green (250 nM, 15 min) and imaged by Hessian-SIM. The representative mitochondrial cristae were displayed. The green arrowhead indicates “cut-through crista”, the red arrowhead indicates “lamellar crista”.

OPA1-Mic10 double knockout (DKO) on mitochondrial EM sections from the cells were analyzed. We observed crista shape. OPA1-Mic10 DKO mitochondria contain that the outer membrane of the onion-like crista is still many onion-like cristae (Fig. S5B), and the number of tethered to the IBM in some continuous sections of the spherical crista was significantly increased in OPA1-Mic10 OPA1-Mic10 DKO EM sample (Fig. S6), but before or DKO mitochondria compared with control or OPA1 KO after these sections, the onion-like crista is completely mitochondria (Fig. 6D–E and Supplementary Videos 21– detached from the IBM (Fig. S6). Further 3D tomographic 22). Moreover, the spherical crista could contact and fuse reconstruction showed that the outer membrane of the with each other in OPA1-Mic10 DKO mitochondria (Fig. spherical crista partly connects to the IBM (Fig. 6G, H, 6D and Supplementary Video 16), suggesting that OPA1 is and Supplementary Videos 17–19), suggesting that this not required for mitochondrial crista membrane fusion. spherical crista is in its final stages of formation and has Furthermore, in addition to the spherical crista, a few cut- not yet completely detached from the IBM. Additionally, through cristae also existed in OPA1-Mic10 DKO mito- Hessian-SIM imaging showed that the spherical crista was chondria (Figs. S5C–S5D). These findings indicate that observed in living OPA1 KO and Mic10 KO cells (Fig. cut-through crista are probably preforms of spherical 7A–C). We then used time-lapse Hessian-SIM to track crista. Because spherical crista is detected in Mic10 KO the process of spherical crista formation in living cells. cells, which still display normal tubular mitochondria (Fig. Some small spherical mitochondria fused with and 4H), the formation of spherical crista is not the con- entered the large spherical mitochondrion to form sphe- sequence of mitochondrial fragmentation. rical crista in Mic10 KO cells (Fig. 7D, E and Supple- Therefore, we propose the following mechanism of spherical mentary Videos 20–21). These findings strongly confirm crista formation: after outer membrane fusion between a large the mode of spherical crista formation (Fig. 6F). and a small mitochondrion, the small spherical membrane Although few mitochondrial spherical cristae are coming from the inner membrane of the small mitochondrion observed in normal HeLa and HCT116 cells, onion-like is sequestered by the IBM of the large mitochondrion and mitochondrial cristae (2D form of spherical crista) are then detached from the IBM to form a spherical crista, whose frequently observed in the mitochondria of cells with membranes arise from two originally distinct inner mito- mtDNA mutations, deletion or loss38,39. We then inves- chondrial membranes (Fig. 6F). During this process, the defi- tigated mitochondrial crista morphology in 143B Rho0 ciency in inner mitochondrial membrane fusion and impaired (ρ0) cells lacking mtDNA. 143B Rho0 mitochondria dis- MICOS activity mainly contribute to the formation of sphe- played remarkably increased onion-like mitochondrial rical crista. cristae (Figs. S7A–S7C), indicating that mtDNA loss To verify our hypothesis, we attempted to capture TEM might be involved in the formation of spherical crista. images of key steps (steps “c” and “d”) during the for- In addition, OPA1 KO led to a dramatic reduction in mation of spherical crista. The continuous 10-nm-thick MICOS components (Mic60, Mic10, Mic13, and Mic19)

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Fig. 6 (See legend on next page.)

and mitochondrial respiratory complex components and Mic19) or mitochondrial respiratory complex com- (SDHA, COX2, and COX4) (Fig. S8A). However, Mic10 ponents (SDHA, COX2, and COX4) (Fig. S8B), indicating KO did not affect MICOS components (Mic60, Mic10, that the spherical crista in Mic10 KO mitochondria still

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(see figure on previous page) Fig. 6 Identification of mitochondrial “spherical crista”.ARepresentative FIB-SEM image of mitochondria in HeLa Mic10 KO cells (left panel). 3D reconstruction and segmentation of mitochondrion showing spherical crista from FIB-SEM were completed by using 3D-IMOD software. White, mitochondrial outer membrane; cyan, mitochondrial IBM; purple red, the spherical crista. B Quantification of the “spherical crista” in mitochondria of control and Mic10 KO HeLa cells. “others” means “other types of crista except for spherical crista” (n = 100 cristae). Statistical significance was assessed from the student’s t-test; error bars indicate the means ± SD of three independent experiments, ***p < 0.001 versus control. C The specimen from HCT116 OPA1 KO cells was analyzed and imaged by FIB-SEM. 3D reconstruction and segmentation of mitochondrion containing spherical crista were performed by using 3D-IMOD software. White, mitochondrial outer membrane; cyan, IBM; purple red, the spherical crista. D The samples of HCT116 Mic10-OPA1 DKO cells were analyzed and imaged by FIB-SEM. 3D reconstruction and segmentation of mitochondrion showing two contacted spherical cristae were completed using 3D-IMOD software. White, mitochondrial outer membrane; cyan, mitochondrial IBM; purple red, the spherical crista. E Quantification of the spherical crista in mitochondria of control, OPA1 KO, and Mic10-OPA1 double knockout (DKO) HCT116 cells (n = 100 cristae). “others” means “other types of crista except for spherical crista”. Statistical significance was assessed from the student’s t-test; error bars indicate the means ± SD of three independent experiments, ***p < 0.001 versus control. F The mode of the spherical crista formation. Two mitochondria contact and process mitochondrial outer membrane fusion, the small mitochondrion is then engulfed into the bigger mitochondria, and detaches from the IBM of the bigger mitochondrion to form the spherical crista. G, H Representative FIB-SEM images of mitochondria (G, H)in HCT116 Mic10-OPA1 DKO cells. 3D reconstruction and segmentation of mitochondrion showing the spherical crista were completed using 3D-IMOD software. White, mitochondrial outer membrane; cyan, mitochondrial IBM; purple red, the spherical crista.

contain normal crista proteins, including respiratory to a re-examination of previous concepts about mito- complexes and MICOS components. Moreover, OPA1 chondrial membrane organization and biogenesis. KO but not Mic10 KO exhibited a decreased mitochon- We discovered two novel types of mitochondrial cristae, drial membrane potential (Figs. S8C–S8D), suggesting cut-through crista and spherical crista (Figs. 3–5). Further- that spherical crista in Mic10 KO mitochondria maintain more, we demonstrated the pathways of cut-through crista a normal mitochondrial membrane potential. and spherical crista formation and proposed the modes of formation of these two types of mitochondrial cristae (Fig. 6F Discussion and S4D). OPA1 dysfunction-mediated inhibition of inner Mitochondrial cristae are the main sites of electron mitochondrial membrane fusion contributes to the forma- transfer and oxidative phosphorylation in mitochondria. tion of these 2 types of cristae. Spherical crista mainly existed The study of mitochondrial crista dynamics and remo- in OPA1- or MICOS-depleted mitochondria and largely deling has been a focus and a challenge in the field of formed in OPA1-Mic10 DKO mitochondria (Fig. 6A–E), but mitochondrial research. The traditional TEM technique no spherical crista were found in normal mitochondria (Figs. can only display the crista morphology of a certain section 6B, E), suggesting that spherical crista are highly associated of a mitochondrial sample and cannot reflect the dynamic with mitochondrial dysfunction. Spherical crista appear as changes of mitochondrial cristae in living cells. Our newly onion-like structures in 2D EM imaging (Figs. S5A and S5B). established Hessian-SIM technique can clearly present the Onion-like cristae were largely formed in response to inhi- mitochondrial crista morphology in living cells and track bition of oligomerization of F1FO ATP synthase40,butoli- the crista’s dynamic change and formation process by gomerization of F1FO ATP synthase is required for the time lapse (Fig. 1 and Supplementary Video 1). However, formation of tubular cristae6, indicating that the model of due to the limitation of its optic resolution, Hessian-SIM spherical crista formation is different from that of tubular cannot distinguish the fusion or contact between mito- cristae formation. Indeed, the model of spherical crista for- chondrial cristae and that between cristae and the IBM. mation is totally different from the “invagination model” and Therefore, we used Hessian-SIM combined with TEM, the “balloon model” (Fig. 6F). Our findings reveal the FIB-SEM and 3D tomographic reconstruction to study diversity of mitochondrial crista morphology and multiple mitochondrial crista dynamics. These technologies com- pathways for crista formation. plement each other functionally: Hessian-SIM enables Mitochondrial crista remodeling is highly associated with visualization of the mitochondrial crista dynamic process mitochondrial functions. Mitochondrial crista shape reg- in living cells, TEM has the advantage of optical resolu- ulates the organization and function of the oxidative phos- tion in visualizing the mitochondrial crista ultrastructure phorylation system, directly affecting cellular energy but can only capture fixed samples, and FIB-SEM and 3D metabolism3,4. In addition, mitochondrial crista remodeling tomographic reconstruction enables the stereoscopic regulates cytochrome c release during apoptosis10,13,andwe presentation of mitochondrial cristae. Therefore, the also observed that mitochondrial crista shape is remodeled in combination of these three techniques is the best choice ActD-treated cells (Fig. S3C–S3D). Moreover, OPA1- for studying mitochondrial crista dynamics and will lead dependent mitochondrial crista remodeling is required for

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Fig. 7 The formation of mitochondrial “spherical crista”.A–C Live HeLa Mic10 KO (A), COS7 Mic10 KO (B), and OPA1 KO MEFs (C) cells were stained with MitoTracker Green (250 nM, 15 min), and then imaged by Hessian-SIM. The red arrowhead indicates the spherical crista. D Representative live-cell time-lapse images of mitochondria in COS7 Mic10 KO cells stained with MitoTracker Green (250 nM, 15 min). Time-lapse Hessian-SIM images reveal that two small mitochondria (the green and red arrowheads indicated) underwent the process of entering into a large mitochondrion. E Representative live-cell time-lapse Hessian-SIM images of mitochondria in COS7 Mic10 KO cells stained with MitoTracker Green (250 nM, 15 min). Time-lapse Hessian-SIM images reveal that a small mitochondrion (the red arrowhead indicated) entered into a giant mitochondrion to form a spherical crista. cellular adaptation to metabolic demand and is critical for 100 U/mL penicillin (Gibco) and 100 mg/mL streptomycin 9 cellsurvivalandgrowth. (Gibco). Cells were maintained in 5% CO2/ 95% air at 37 °C. For Hypoxia exposure, cells were placed in a modulator Materials and methods chamber with 1% O2 /5% CO2 /94% N2 at 37 °C. Lipo- Cell culture and reagents fectamine 2000 (Invitrogen) were carried out as protocol HeLa, HCT116, MEFs, and COS7 cells were cultured in present. MitoTracker Green and MitoTrakcer Red used for high-glucose DMEM supplemented with 10% FBS (PAN), Hessian-SIM imaging were obtained from Invitrogen.

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Hessian structured illumination microscopy (Hessian-SIM) cristae retaining more than two seconds (excluding false imaging events due to imaging angle), it is called “cristae division”. Hessian-SIM analysis was performed as described pre- Due to the limitation of SIM resolution, only the contact, viously27. Cells used in this study were seeded onto poly- fusion, division, or detachment events retained for more L-lysine-coated coverslips, and cultured for an additional than 2 s (excluding false events due to imaging angle) 12–18 h before the SIM imaging. To label the mito- were counted. All quantifications were analyzed by three chondria, cells were incubated with 250 nM MitoTracker independent experiments (at least 100 mitochondrial Green FM (Thermo Fisher Scientific) in high-glucose cristae in 10–30 mitochondria from 10–20 cells were DMEM at 37 °C for 15 min, and then washed with HBSS analyzed for each experiment or each group). The dif- + + solution containing Ca2 ,Mg2 but no phenol red ference between groups was compared by the student’s t- (Thermo Fisher Scientific) three times. Super-resolution test, the asterisks *, **, and *** denote statistical sig- images acquired on a Hessian-SIM microscope equipped nificance with P < 0.05, 0.01, and 0.001, respectively. with a TIRF objective (ApoN 100X/1.7 HI Oil, Olympus) and a multiband dichroic mirror (DM, ZT405/488/561/ Transmission electron microscopy (TEM) analysis 640-phase R; Chroma), and three lasers (Sapphire488LP- The procedure of TEM was performed according to the 200, Coherent; Sapphire 561LP-200, Coherent; and MRL- previous report41. HeLa, HCT116, and 143B cells were III -640-150, IL photonics) as the light sources, and an fixed with 3% PFA, 0.5% glutaraldehyde, and 0.25% acoustic optical tunable filter (AOTF, AA Opto-Electro- sucrose in 0.1 M sodium phosphate buffer (PH 7.4) for 1 h nic, France) to combine, switch, and adjust the illumina- at RT, then were washed with 0.1 M PB contain 0.25% tion power of the lasers. Nine raw frames illuminated with sucrose (PH 7.4). Next, cells were collected using a cell a periodic pattern of parallel lines and shifted through scraper and subsequently were gradient centrifuged, the three phases for each of three orientation angles were samples were then postfixed with 2% osmium tetroxide in used to reconstruct one SR image. The exposure time was 0.1 M PB at 4 °C for 1 h, 2% uranyl acetate overnight 4 °C. set to 10 ms for each raw images capture. All SIM images Fixed samples were dehydrated with different con- were analyzed with the Hessian-SIM microscopy method centrations of ethanol, embedded in Questol-812, and and the ImageJ software. Four ImageJ plugins were used polymerized at 60 °C for 48–72 h. The blocks were to improve the SIM images quality. ultrathin-sectioned at 70 nm with a diamond knife using an ultramicrotome (Leica). Sections were placed on cop- Mitochondrial cristae morphology and dynamics analysis per grids and stained with 2% Lead citrate at RT for Cells were stained with MitoTracker Green FM 15 min and then rinsed with the distilled water. The grids (Thermo Fisher Scientific) to label mitochondrial cristae were observed and imaged under a JEM-1400Plus (JEOL) and inner boundary membrane (IBM), and different transmission electron microscope at an acceleration vol- mitochondrial cristae dynamic events were quantified tage of 80 kV. manually by observing the corresponding Live-Hessian- SIM movies for a time span of 0–10 s at a frame rate of Focused ion beam/scanning electron microscopy (FIB-SEM) 0.1 s/frame. The number of “Shortening” and “Elongation” analysis events are calculated by the cristae length, each crista that HeLa and HCT116 cells were fixed, filtrated, and is shortened or elongated beyond 50 nm is considered a polymerized as described previously28. Focused ion beam dynamic event. The number of “Crista–IBM contact” milling and scanning electron microscopy imaging were (mitochondrial crista contact with the inner boundary carried out with an FEI Helios NanoLab G3 UC (from membrane) and “Crista–Crista contact” (mitochondrial Tsinghua, China). FIB milling was performed with 600 pA crista contact with another crista) events were observed to 20 nA for the given samples. SEM‐Imaging current was by continuously cristae change, and the “Crista–IBM 0.4 nA. FIB milling steps was 10 nm/slice, and each slice contact” or “Crista–Crista contact” events retaining over was imaged. Accordingly, each image represents 10 nm of 2 s (excluding false events due to imaging angle) were the stack, at ×35,000 magnification. The selected mito- recorded and counted. The intact mitochondrial crista chondria were there-dimensional (3D) reconstituted and containing one or two crista junctions is separated from 3D rendered by IMOD software. the inner boundary membrane to form a new crista losing one crista junction, this event is identified as “Cristae SDS-PAGE and western blotting analysis detaching from the IBM”. The event in two separate Cells were harvested by 0.25% trypsin (Gbico), cell mitochondrial cristae contact and merge into a single suspension was then centrifuged, pellet cells were resus- crista that retained for more than two seconds (excluding pended with 1× sample buffer, and boiled for 12 min. The false events due to imaging angle), is called “cristae protein samples were separated by SDS-PAGE and fusion”. If one mitochondrial crista is divided into two transferred into 0.45 μm PVDF membranes, 2 h later,

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